|Home | About | Journals | Submit | Contact Us | Français|
A peptide from cadherin AgCad1 of Anopheles gambiae larvae was reported as a synergist of Bacillus thuringiensis subsp. israelensis Cry4Ba's toxicity to the Anopheles mosquito (G. Hua, R. Zhang, M. A. Abdullah, and M. J. Adang, Biochemistry 47:5101-5110, 2008). We report that CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) and a longer peptide, CR9 to CR11 (CR9-11), from AgCad1 act as synergists of Cry4Ba's toxicity to Aedes aegypti larvae, but a Diabrotica virgifera virgifera cadherin-based synergist of Cry3 (Y. Park, M. A. F. Abdullah, M. D. Taylor, K. Rahman, and M. J. Adang, Appl. Environ. Microbiol. 75:3086-3092, 2009) did not affect Cry4Ba's toxicity. Peptides CR9-11 and CR11-MPED bound Cry4Ba with high affinity (13 nM and 23 nM, respectively) and inhibited Cry4Ba binding to the larval A. aegypti brush border membrane. The longer CR9-11 fragment was more potent than CR11-MPED in enhancing Cry4Ba against A. aegypti.
Mosquitoes are vectors of human and animal infectious diseases. Aedes (Stegomyia) aegypti can transmit viruses that cause dengue fever and yellow fever. Mosquitoes have shown a rapid increase in resistance to various chemical insecticides (16). Nonchemical larvicides based on the bacterium Bacillus thuringiensis subsp. israelensis de Barjac are used to control mosquitoes. The specific toxicity of B. thuringiensis subsp. israelensis to Anopheles, Culex, and Aedes spp. is due to the protein components of the parasporal crystal (reviewed in reference 9). The Cry4Ba insecticidal protein is one of at least four types of parasporal crystals expressed in B. thuringiensis subsp. israelensis. The Cry4Ba insecticidal protein is highly toxic to Anopheles and Aedes larvae but not to Culex larvae (2, 6).
Synergists of B. thuringiensis subsp. israelensis, another strategy to improve the efficacy of Cry4Ba and B. thuringiensis subsp. israelensis, would lead to the reduced quantity needed to obtain control, lengthen residual activity, and possibly delay the onset of resistance in target insects (7, 8, 10, 21). In the case of mosquitocidal Cry11Aa, synergistic cytolytic toxin functions as an adventitious receptor, inducing prepore formation and subsequent membrane insertion (20). Recently, a new type of synergist based on peptide fragments of host insect cadherins was shown to enhance Cry1A, Cry3, and Cry4Ba toxicities to lepidopteran, coleopteran, and dipteran larvae, respectively (5, 11, 18, 19). A fragment of the Anopheles gambiae larva midgut cadherin AgCad1 was shown to enhance Cry4Ba against A. gambiae (11). Here we show that the C-terminal cadherin repeat (CR) CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) of AgCad1 and another fragment (CR9 to CR11 [CR9-11]) also enhance Cry4Ba against another important mosquito species, A. aegypti.
The CR9-11 and CR11-MPED regions of AgCad1 were overexpressed in Escherichia coli according to Chen et al. (5) and tested for the ability to enhance Cry4Ba toxicity to A. aegypti larvae. The CR11-MPED plasmid has been described previously (11), and CR9-11 in pET30a was constructed using the same method, with primers 5′-CGA GCA TAT GGG GTC CCC G TT GCC GAA ATT and 5′-CGC TCT CGA GAA ACA C GA ACG TCA CGC GGT TC. To determine the extent that CR9-11 and CR11-MPED could enhance a low dose of Cry4Ba inclusion body form (IBF), we added increasing amounts of CR9-11 and CR11-MPED IBFs to a Cry4Ba IBF concentration predicted to cause about 35% larval mortality. Bioassays were conducted with fourth-instar A. aegypti larvae as previously described (11). Each treatment was replicated four times, each replicate contained 10 larvae, and larval mortality was recorded after 16 h. The enhancement effect reached a plateau at a 1:25 (Cry4Ba/peptide) mass ratio for both AgCad1 fragments (data not shown). To determine the specificity of the cadherin effect, we included the partial cadherin-like protein WCR8 to WCR10 (WCR8-10) from western corn rootworm Diabrotica virgifera virgifera (18), using a Cry4Ba/WCR8-10 mass ratio of 1:100. The control bioassay using the WCR8-10 cadherin fragment from D. virgifera virgifera showed no synergistic effect with Cry4Ba (data not shown).
To assess the relative increase in toxicity when cadherin fragments were present, larvae were fed the Cry4Ba IBF alone or with a fixed 1:25 mass ratio of AgCad1 peptide. The calculated 50% lethal concentration (LC50) of the Cry4Ba IBF was 20.34 ng/ml (16.37 to 25.93 ng/ml) (Table (Table1).1). The addition of CR9-11 and CR11-MPED IBFs to Cry4Ba IBF reduced the Cry4Ba LC50s to 3.43 ng/ml (1.66 to 5.80 ng/ml) and 7.35 (5.94 to 9.07 ng/ml), respectively (Table (Table1);1); furthermore, soluble forms (SF) of CR9-11 and CR11-MPED also reduced the Cry4Ba IBF LC50s, to 5.79 ng/ml (4.42 to 6.73 ng/ml) and 9.23 ng/ml (7.53 to 11.33 ng/ml), respectively (Table (Table1).1). The increased synergistic levels of longer cadherin fragments that are involved with toxin binding were also observed with cadherin fragments from Manduca sexta (3). The use of the SF led to a lower level of enhancement than those of the IBFs of the cadherin peptides. This might be explained by the fact that mosquito larvae are filter feeders; thus, more peptides are ingested if they can be filtered by the mosquito (22).
The binding affinity between Cry4Ba and CR9-11, CR11-MPED, or WCR8-10 was determined with microtiter plates and an enzyme-linked immunosorbent assay, as described previously (24). Microtiter plates were coated with 1.0 μg Cry4Ba toxin/well. Biotinylated CR9-11 and CR11-MPED (0.001 nM to 100 nM) were used to determine total binding values. As shown in Fig. Fig.1,1, each biotin-labeled cadherin peptide specifically bound Cry4Ba toxin. Using a one-site saturation model, we calculated Kd (dissociation constant) values for cadherin peptide binding to Cry4Ba toxin, as follows: CR9-11 peptide Kd value of 13.3 ± 2.4 nM, CR11-MPED peptide Kd value of 23.2 ± 3.4 nM, and WCR8-10 Kd value of 30.0 ± 6.6 nM. Results from these assays are evidence of a specific and high-affinity interaction between Cry4Ba and the two AgCad1 fragments. However, the high-affinity binding of Cry4Ba to WCR8-10 was unexpected, since the cadherin fragment did not affect Cry4Ba toxicity.
AgCad1 CR peptides reduce Cry4Ba binding to brush border membrane vesicles (BBMV). Using unlabeled cadherin peptides and Cry4Ba toxin as competitors, we performed competition binding experiments using 125I-Cry4Ba and A. aegypti BBMV, as described by Jurat-Fuentes and Adang (13), with slight modifications (24). Samples were used in duplicate, binding experiments were repeated, and the averaged data were used for analysis. Unlabeled Cry4Ba competed against 125I-Cry4Ba binding to BBMV from about 13.5 to 10 pmol toxins bound per μg BBMV (Fig. (Fig.2).2). AgCad1 CR peptides, but not WCR8-10, reduced binding to the same extent and at the same competitor concentrations (in nM) as unlabeled Cry4Ba. Although WCR8-10 binds Cry4Ba with high affinity (Kd = 30 nM), the inability of WCR8-10 to compete against Cry4Ba binding to A. aegypti BBMV suggests that it did not share the same binding sites as the AgCad1 CR peptides. The differences in the binding characteristics of these cadherin fragments could be responsible for the different levels of synergistic effects that were observed.
How can a cadherin fragment inhibit Cry toxin binding to BBMV yet synergize Cry toxicity to larvae? One explanation is that AgCad1 is not a receptor for Cry4Ba in A. gambiae larvae, as we suggested previously (11), and that its orthologue is not a receptor in A. aegypti. Possibly, AgCad1 is a “null” receptor for Cry4Ba that does not mediate toxicity, and by blocking Cry4Ba binding to cadherin, the toxicity to larvae is increased. The concept of null receptors was proposed to account for Cry1A binding proteins in the midguts of lepidopteran larvae that do not correlate with toxicity (14). Another explanation is that AgCad1 CR peptides bind Cry4Ba, inducing prepore formation and subsequent binding to secondary receptors, similarly to Cry1Ab, which forms a prepore structure that binds aminopeptidase, a secondary receptor in M. sexta (4). Studies show that M. sexta synergist CR12-MPED binds Cry1Ab with high affinity (5) and induces Cry1Ab oligomerization in the presence of midgut proteinases or trypsin (23). Recently, a Helicoverpa armigera cadherin fragment was shown to oligomerize and enhance the toxicity of Cry1Ac (19). The toxin oligomerization step was reported to be necessary for toxicity (12) and was shown to correlate with enhancement activity of toxin-binding cadherin fragments (17). However, the correlation between toxin enhancement and toxin oligomerization was inconsistent, as a toxin-binding cadherin fragment that oligomerizes Cry1Ac was shown to reduce toxicity (15). Further research is necessary to establish the mechanism of AgCad1 CR peptide synergism of Cry4Ba toxicity to A. gambiae (11) and A. aegypti larvae.
This research was partially supported by National Institutes of Health grant R01 AI 29092.
We thank Milton Taylor (InsectiGen, Inc.) for helpful suggestions during the course of this study and for reviewing a version of the manuscript. Krishna Bayyareddy provided valuable technical assistance.
Published ahead of print on 2 October 2009.